1. Field of the Invention
The present invention relates to rapid determination of the infection and food contamination with Listeria monocytogenes, and more particularly to a monoclonal antibody binding specifically to the p60 protein of Listeria monocytogenes, a hybridoma cell producing the monoclonal antibody, a test kit comprising the monoclonal antibody, and a method for detecting Listeria monocytogenes using the monoclonal antibody.
2. Background of the Related Art
Listeria species are Gram-positive, facultative rods which are widely distributed in the natural environment. Listeria spp. includes Listeria monocytogenes, Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri and Listeria grayi. Among them, only Listeria monocytogenes is pathogenic to humans, and strains pathogenic to animals are Listeria monocytogenes and Listeria ivanovii. Particularly, if Listeria monocytogenes contaminates food requiring cold storage, it can survive and proliferate in the low-temperature storage process for a long time period so that it becomes an important bacillus causing food poisoning mediated by cold storage food. Furthermore, if listeriosis caused by this bacillus becomes serious, it will cause septicemia and meningitis and induce miscarriage of pregnant women. Listeria monocytogenes is particularly hazardous to infants, very old persons, pregnant women and immune deficient patients, and shows a high death rate reaching 30% in patients with listeriosis. This listeriosis is known to be caused mainly by foods, such as meat, meat processing products, vegetables, and milk processing products. Thus, rapid diagnosis of the food contamination with Listeria monocytogenes is necessary for the prevention of listeriosis.
Listeria identification methods which can be currently used in hospitals, industries (food companies, meat companies and milk processing companies) and the like include various conventional methods using biochemical assays, but they are inefficient methods requiring much time and labor.
Diagnostic methods which are used for the rapid determination of the infection and food contamination with Listeria monocytogenes include molecular biological methods, such as polymerase chain reaction (PCR), but such methods are unsuitable for actual clinical and industrial applications due to limitations in technical capabilities and accuracies. Moreover, immunological methods such as enzyme immunoassay (EIA) are frequently used, but show non-specificity, which leads to inaccurate test results or makes additional verification tests necessary.
Kits known to be used for the rapid detection of Listeria bacteria include non-radioactive DNA probe kits using PCR techniques and nucleic acid hybridization assays, and kits using immunoassays. Furthermore, there are kit products developed for a dipstick assay using immunochromatography. However, all the kits have a disadvantage in that they either cannot specifically detect only Listeria monocytogenes or detect all Listeria species.
Since it was difficult to produce a monoclonal antibody to Listeria monocytogenes, an ELISA or dipstick detection kit with a monoclonal antibody which selectively recognizes this bacterial strain could not be developed. This is because either the whole Listeria monocytogenes strain was used as an antigen, or even when certain antigens were used, a monoclonal antibody specific only to Listeria monocytogenes was not produced.
Murein hydrolase p60 proteins which exist commonly in Listeria spp. are exo enzymes that Listeria secretes for cell division. Prior to the present invention, polyclonal antibodies were produced using certain peptides of p60, for example, pepA and pepD (Bubert A. et al., Appl. Environ Microbiol, 60(9), 3120-7, 1994). However, such polyclonal antibodies showed weak effects due to their low titer and the unsuitable application of a sandwich ELISA system, so that they were insufficient for commercial use. The present inventors have produced a new monoclonal antibody which can selectively recognize Listeria monocytogenes whose putative epitope may differ from the pepA or pepD peptide region of p60 that has been described somewhere (Example 4 and FIG. 4a).
Under this background, the present inventors have produce a monoclonal antibody recognizing a specific epitope of Listeria monocytogenes p60 proteins, among murein hydrolase p60 proteins which exist commonly in Listeria genus.